Method of detecting colon cancer

ABSTRACT

An immunological assay and kit for colon cancer screening is disclosed. Fecal glycoproteins are extracted from individual samples such that immunogenicity is maintained. The purified fecal glycoproteins are reacted with antibodies to Colon and Ovarian Tumor Antigen (COTA). The mucin antigen COTA is specifically present in colorectal cancer tissue and not in normal colons. The amount of COTA in the fecal sample is determined and used to indicate the presence of colon cancer.

FIELD OF THE INVENTION

[0001] The present invention is directed to a method for detecting coloncancer utilizing an antibody specific for a glycoprotein found in coloncancer cells.

BACKGROUND OF THE INVENTION

[0002] Colorectal cancer is among the leading causes of cancer-relatedmorbidity and mortality in industrialized nations. The pathogenesis isrelated to hereditary influences, modified by the quantity and qualityof dietary fat. In 1995, the American Cancer society estimated that135,000 new cases of colon cancer were diagnosed; 71% were in the colonand 30% were in the rectum. Patients diagnosed at an early stage, priorto lymph-node spread, are potentially cured with surgery. At present,only 41% of patients are diagnosed at an early stage. The remainingcases frequently undergo peri-operative radiation and/or chemotherapy toattempt to control the metastatic spread of disease. Ultimately, 50% ofpatients thought to have undergone curative resections eventuallydevelop recurrent disease. Unfortunately, 55,000 Americans die each yeardue to recurrent or metastatic colon or rectal cancer. The key toenhanced survival is early diagnosis. Colon and rectal cancers are oftensilent and slowly progressive. Most patients exhibit symptoms such asrectal bleeding, pain, abdominal distension or weight loss only afterthe disease is advanced and not surgically curable.

[0003] Over the past 25 years, early colorectal cancer detection hasbeen based on the fecal occult blood test (FOBT) performed annually onasymptomatic individuals. Current recommendations adapted by severalhealthcare organizations, including the American Cancer Society, callfor fecal occult blood testing beginning at age 50, repeated annuallyuntil such time as the patient would no longer benefit from screening. Apositive FOBT leads to colonoscopic examination of the bowel; anexpensive and invasive procedure, with a serious complication rate ofone per 5,000 examinations. Only 12% of patients with heme positivestool are diagnosed with cancer or large polyps at the time ofcolonoscopy. Most studies show that FOBT screening does not improvecancer-related mortality or overall survival. Compliance with occultblood testing has been poor; less than 20 percent of the population isoffered or completes FOBT as recommended. If FOBT is properly done, thepatient collects a fecal sample from three consecutive bowel movements.Samples are obtained while the patient adheres to dietary guidelines andavoids medications known to induce occult gastrointestinal bleeding. Inreality, physicians frequently fail to instruct patients properly,patients frequently fail to adhere to protocol, and some patients findthe task of collecting fecal samples difficult or unpleasant, hencecompliance with annual occult blood testing is poor. Compounding theproblem of compliance, the sensitivity and specificity of FOBT to detectcolon cancer is poor. In eight prospective studies where hemocculttesting was followed by colonoscopy, only 41 of 159 cancers diagnosedwere detected by FOBT, yielding a screening sensitivity of 26%. Fobtsensitivity for pre-cancerous polyps was also poor. Poor testspecificity leads to unnecessary colonoscopy, adding considerableexpense to colon cancer screening. In the University of Minnesota trial,a large prospective hemoccult screening study, test specificity was 90%,and positive predictive value was 2%. Only one colon cancer was found inevery 50 test-triggered colonoscopies performed.

[0004] New methodology of immunological testing has potential advantagesover FOBT including improved sensitivity, specificity and patientcompliance. If immunological testing is more sensitive and specific thanFOBT, the frequency of testing could be reduced, collection ofconsecutive samples would be eliminated, dietary and medication schedulemodifications would be eliminated, and patient compliance would beenhanced. If colon cancer screening by immunological testing is morespecific, the problem of false positive test results leading tounnecessary colonoscopic examination would be reduced leading to costsavings and improved safety. Clearly, there is a long felt need for asimple, accurate, and inexpensive screen for colon cancer.

DESCRIPTION OF THE RELATED ART

[0005] Since the goblet cells of colorectal cancers produce glycoproteinmucin(s) that are immunologically distinguishable from normal colonicmucin (Nairn et al. Br. Med. J. 1791-1793, 1962) it is possible todetect their presence in the feces by immunological assay. Springer(Springer Science 1198-1206, 1984) reported that T antigens (ThomsenFriedenreich), and Tn antigens, precursors of MN blood groupglycoproteins, are tumor associated antigens. Kurosaka et al (Kurosakaet al J. Biol. Chem. 258: 11594-11598, 1983) isolated severaloligosaccharides from a rectal adenocarcinoma and reported that one ofthe major oligosaccharides was sialylated-Tn or STn (NeuAcα2-6GalNAcα1-o-ser/thr). Ovine submaxillary mucin is identical in chemicalstructure as STn disaccharides. Kjeldsen et al. (Kjeldsen, et al. CancerRes. 48: 2214-2220, 1988) produced TKH1 and TKH2 monoclonal antibodiesthat react with ovine submaxillary mucin and demonstrated that both werereactive by immunohistochemistry with adenocarcinoma of human lung,stomach, colon, breast and pancreas, but not with normal human tissuesamples. Itzkowitz et al. (Itzkowitz et al. Cancer Res. 260: 8262-8271,1989) observed expression of all the three Tn, Sialosyl-Tn and Tantigens in colon cancers. He proposed that in malignancy incompleteglycolsylation and early sialylation of precursor antigen results inpremature termination of the carbohydrate chain. In contrast, Podolsky(Podolsky J. Bio. Chem. 260: 15510-15515, 1985) had isolated 21oligosaccharides from normal colonic mucin and sialylated-Tn was one ofthem. The non-reactivity of TKH2 antibody with normal colonic mucosa wasexplained by the work of Jass et al. (Jass et al. Pathology 26: 418-422,1994) and later Ogata et al. (Ogata et al. Cancer Res. 55: 1869-1874,1995) who reported that in the normal colon sialic acid is heavilyo-acetylated and is masked, thus antibodies cannot react, while inneoplastic tissue o-acetylation is not extensive, and sialic acid is notmasked. Removal of the O-acetyl groups from normal colon tissue byalkaline treatment made them reactive with TKH2 antibody.

[0006] Pant et al. did initial studies on Colon-Ovarian tumor antigen(COTA) with polyclonal antibodies produced against mucin extracted fromhuman colon cancer tissue removed at surgery. The polyclonal antibodieswere made specific by absorption with lyophilized extracts of normalhuman colon and other normal human tissues and CEA. The resultantantibody retained immunoreactivity towards colon cancer and mucinousovarian tumors but did not react with normal colon tissue as seen byimmunodiffusion and immunofluorescence testing (Pant et al. Tumor Biol.5: 243-254, 1984). Furthermore, the absorbed anti-COTA antibodiesimmunostained several colon cancer tissues and LS174T tumor cells andother colon cancer xenografts but did not stain normal human colonsections (Pant et al. Am J. Clin. Path. 86:1-9, 1986). Pant used COTAisolated from LS174T tumor cells to produce a monoclonal antibody SP-21.This antibody gave identical immunohistochemical staining pattern asobserved with absorbed polyclonal anti-COTA antibodies (Pant et al.Hybridoma 5: 129-135, 1986). In an extended immunohistochemical studywith SP-21 monoclonal antibody, Dorman et al (Dorman et al. J. Clin.Path. 45: 932-933, 1992) observed that SP-21 immunostained several otherhuman cancer tissues including ovary, stomach, breast, esophagus,prostate, pancreas and endometrium.

[0007] The relationship of COTA to STn was established by chemicalanalysis of purified COTA, blocking of immunoreactivity by N-acetylneuraminic acid and loss of immunoreactivity after neuraminidasetreatment. It was concluded that neuraminic acid is an essentialcomponent of COTA (Pant, et al. Journal of Tumor Marker Oncology 3:1-13, 1988). The identity of COTA to STn was further established bycomparison of SP-21 with TKH1. Kordari et al. reported neuraminidasetreatment and O-glycanase treatments of colon cancer tissue completelydestroyed TKH1 and COTA epitope reactivity indicating that TKH1 and COTAmonoclonal antibodies recognized the NeuAcα2-6GalNAc disaccharideexclusively (Kordari, et al. Tumor Biol. Abs., 1990).

SUMMARY OF THE INVENTION

[0008] The present disclosure is drawn to a method for extractingglycoproteins from a fecal sample such that immunogenicity is maintainedcomprising the steps of:

[0009] (a) obtaining a fecal sample from an individual;

[0010] (b) shaking the fecal sample in a preservative solution;

[0011] (c) separating the solution containing the fecal sample toproduce a fraction comprising glycoproteins;

[0012] (d) precipitating the glycoproteins from the fraction comprisingglycoproteins; and

[0013] (e) dissolving the precipitated glycoproteins in buffer.

[0014] The method may further comprise the steps of:

[0015] (f) centrifuging the solution from step (e) to produce a pelletand a supernatant; and

[0016] (g) collecting the supernatant containing the extractedglycoproteins.

[0017] In a preferred embodiment, the fecal sample is collected in aclean vial containing preservative wherein the preservative comprisesethanol and formalin at a concentration such that bacterial growth isretarded and extraneous fecal matter is precipitated while maintainingimmunogenicity of glycoproteins in the fecal sample. Preferably, thepreservative comprises 25-45% ethanol with 0.025%-0.35% formalin. Morepreferably, the preservative comprises 40% ethanol with 0.25% formalin.

[0018] In a preferred embodiment, the solution containing the fecalsample is separated by centrifugation. More preferably, thecentrifugation is at 1040-1500×g for 10-15 minutes at room temperature.

[0019] In a preferred embodiment, the glycoproteins are precipitatedfrom the fraction comprising glycoproteins with 3 volumes of 100%ethanol with 0.1 ml of 20% sodium acetate. More preferably, theprecipitation proceeds for about 3 hours at room temperature.Preferably, the precipitated glycoproteins are dissolved in phosphatebuffered saline.

[0020] The present disclosure is also drawn to a method for screeningfor colon cancer comprising:

[0021] (a) obtaining purified fecal glycoproteins, said glycoproteinsbeing obtained by a method comprising:

[0022] (i) obtaining a fecal sample from an individual;

[0023] (ii) shaking the fecal sample in a preservative solution;

[0024] (iii) separating the solution containing the fecal sample toproduce a fraction comprising glycoproteins;

[0025] (iv) precipitating the glycoproteins from the fraction comprisingglycoproteins; and

[0026] (v) dissolving the precipitated glycoproteins in buffer; and

[0027] (b) determining the level of COTA antigen in the purified fecalglycoproteins.

[0028] In a preferred embodiment, the fecal sample is collected in aclean vial containing preservative wherein the preservative comprisesethanol and formalin at a concentration such that bacterial growth isretarded and extraneous fecal matter is precipitated while maintainingimmunogenicity of glycoproteins in the fecal sample. Preferably, thepreservative comprises 25-45% ethanol with 0.025%-0.35% formalin. Morepreferably, the preservative comprises 40% ethanol with 0.25% fornalin.

[0029] In a preferred embodiment, the solution containing the fecalsample is separated by centrifugation. More preferably, thecentrifugation is at 1040-1500×g for 10-15 minutes at room temperature.

[0030] In a preferred embodiment, the glycoproteins are precipitatedfrom the fraction comprising glycoproteins with 3 volumes of 100%ethanol with 0.1 ml of 20% sodium acetate. More preferably, theprecipitation proceeds for about 3 hours at room temperature.Preferably, the precipitated glycoproteins are dissolved in phosphatebuffered saline.

[0031] In a preferred embodiment, the determination of the level of COTAantigen in the purified glycoproteins comprises the steps of:

[0032] (a) reacting an antibody for COTA antigen with the extractedglycoproteins to form a complex of the antibody and the COTA antigen;

[0033] (b) exposing the complex to a second antibody, wherein saidsecond antibody is a detection agent; and

[0034] (c) determining the level of the detection agent and in turndetermining the presence of COTA antigen in the fecal sample.

[0035] In one embodiment, the antibody for COTA antigen is bound to asolid surface. In an alternate embodiment, the extracted glycoproteinsare bound to a solid surface. Preferably, the antibody for COTA antigenis monoclonal antibody SP-21.

[0036] The present disclosure is also drawn to a kit for screening forcolon cancer comprising:

[0037] an anti-COTA capture antibody bound to a solid support;

[0038] purified human COTA antigen; and

[0039] a vial containing a preservative solution.

[0040] In one embodiment, the solid support is an ELISA plate. In analternate embodiment, the solid support is a membrane filter. In apreferred embodiment, the kit contains the monoclonal antibody SP-21 asthe antibody for COTA antigen.

[0041] In a preferred embodiment, the kit contains a preservativesolution which comprises 25-45% ethanol with 0.025%-0.35% formalin. In amost preferred embodiment, the kit contains a preservative comprising40% ethanol with 0.25% formalin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1. Plot of optical density of slot-dots obtained on dilutionsof purified COTA protein from LS174T.

[0043]FIG. 2. Lane A. Graded intensity of slot-dots of purified COTAdilutions. Lane B. Slot-dots of 3 patients with colon cancer, 1 withulcerative colitis and dysplasia, 1 with polyp, 3 normal individuals andcontrol PBS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0044] The hemocult test is based on pseudoperoxidase activity of hemewhich oxidizes gum guaiac to a blue color. It is well recognized thatthe test also detects presence of blood in the digestive tract fromsources other than colorectal cancers or adenomas. Furthermore, theperoxidase activity is also present in some foods. Certain chemicalswhich inhibit the pseudoperoxidase enzyme activity can yield falsenegative results affecting the sensitivity and specificity of the test.

[0045] The present disclosure relates to an immunological assay methodand kit to apply to population-based colon cancer screening which avoidsproblems associated with the hemocult test discussed above.

[0046] In a preferred embodiment, the fecal sample is obtained using acommercially available stool collection device such as one availablefrom Ability Building Center, Rochester, Minn., and is then placedimmediately in a preservative solution so as to inhibit bacterialmultiplication and sample glycoprotein degradation. Glycoproteins arethen isolated from the fecal sample at a later time. The preservativesolution contains ethanol and formalin at a concentration such thatbacterial growth is retarded and extraneous fecal matter is precipitatedwhile maintaining immunogenicity of glycoproteins in the fecal sample.Extraneous fecal matter includes components other than the glycoproteinfraction. Preferably, the preservative solution contains 0.025%-0.35%formalin in 25-45% ethanol. In a preferred embodiment, the preservativecontains 40% ethanol with 0.25% formalin to optimally prevent bacterialmultiplication and glycoprotein degradation without loss of glycoproteinimmunoreactivity.

[0047] A key feature of the presently claimed invention is theextraction of the desired glycoproteins from the preservative solutionwhile maintaining immunogenicity. Any method for extraction ofglycoproteins which maintains immunogenicity of the extracted mucins maybe used in the screening method. In general, the fecal sample in thepreservative solution is separated to obtain a fraction containing theglycoproteins. Any means known to those skilled in the art may be usedto fractionate the fecal sample including, but not limited tocentrifugation, filtration, and chromatography. In a preferredembodiment, centrifugation is used to isolate theglycoprotein-containing fraction, preferably at 1040×g to 1500×g for10-15 min. Most preferably, centrifugation at 1040×g for 10 minutes atroom temperature is used to obtain a supernatant containingglycoproteins. The glycoprotein-containing fraction is then furtherpurified by precipitation of the glycoproteins including COTA. Means toprecipitate the glycoproteins include, but are not limited to,precipitation with ammonium sulfate, or organic solvents, such asethanol and acetone, or a combination thereof. Alternatively, theglycoproteins may be further purified by means known to those skilled inthe art such as column chromatography, gel filtration and the like. In apreferred embodiment the glycoprotein-containing fraction isprecipitated with ethanol and sodium acetate and then centrifuged toobtain a precipitate. The precipitated pellet is resuspended in bufferand recentrifuged to obtain a supernatant containing the glycoproteinsto be screened, preferably at 1500×g to 2040×g for 15-20 minutes. In apreferred embodiment, the resuspended pellet is centrifuged at 1500×gfor 15 minutes.

[0048] The level of COTA antigen in the supernatant containing theglycoproteins is then determined. The mucin antigen COTA is specificallypresent in colorectal cancer tissue and not in normal colon tissue.Since COTA mucin is produced by goblet cells and eventually gets mixedwith the feces, detection and estimation of it in the fecal samples isthe basis of the present non-invasive test for colorectal cancer.

[0049] Any antibody which binds to Colon and Ovarian Tumor Antigen(COTA) may be used for quantitation of COTA levels as a colon cancerscreen. A representative monoclonal antibody includes SP-21. Amonoclonal antibody which binds to COTA may be obtained by isolation ofCOTA from the LS174T cell line (ATCC CL-188) using well known methods(Kohler and Milstein (1975) Nature 256: 495-497, for example). Theantibody for COTA antigen is reacted with the extracted glycoproteins toform a complex of the antibody and the COTA antigen. In a preferredembodiment, a combination of one IgG antibody and one IgM antibody isused. Any means available for facilitation of antibody-antigen bindingmay be used in the disclosed method including but not limited to tubes,filters, beads, multiwell plates and a mixture thereof. Preferredembodiments use either ELISA plate technology or slot dot assays. Meansto quantitate the extent of binding include detection using colorimetricassays as well as radioimmunoassay. In a preferred embodiment, thecomplex of the antibody and the COTA antigen is exposed to a secondantibody which is labelled such that the level of COTA antigen in theglycoprotein sample may be detected and quantitated by reference to astandard curve prepared from dilutions of purified COTA. Such labelsinclude, but are not limited to, radioactive and calorimetric methodsincluding absorption, bioluminescence and fluorescence labeling means.In a preferred embodiment, the second antibody is biotinylated and issubsequently treated with peroxidase conjugated streptavidin to producea quantifiable calorimetric signal. Levels of COTA antigen detected bythe Dot assay greater than 15 μg/ml predict the presence of colon cancerwith sensitivity of 83% and specificity of 96%. ELISA methodology mayalso be used to detect COTA antigen. A cut off value for detection ofcolon cancer in μgm/ml will be based upon values obtained from normalindividuals.

[0050] The invention is further set forth in the following examples,which are in no way intended as a limitation upon the scope of theinvention.

EXAMPLES

[0051] A more detailed description of the present invention is providedbelow. While the described embodiment represents the preferredembodiment of the present invention, it is to be understood thatmodifications will occur to those skilled in the art without departingfrom the spirit of the invention. The scope of the invention istherefore to be determined solely by the appended claims.

Example 1 Patient Selection and Sample Collection

[0052] One hundred patients with risk factors or signs of colon cancerscheduled for colonoscopic examination were contacted. Indications forcolonoscopy included hemoccult positive stools, rectal bleeding, priorhistory of adenoma or colon cancer, or family history of colon cancer. Akit containing a vial with 20 ml of preservative (40% ethanol with 0.25%formalin solution) and a fecal collection device were mailed to thepatient. Patients were asked to collect approximately 1-2 gm of fecesand place it into the vial. The kit was returned on the day of theircolonoscopy. Extraction of glycoproteins from fecal samples

[0053] The contents of the vial were shaken vigorously to mix the fecalsample completely and then centrifuged at 1040×g for 10 minutes at roomtemperature (RT) to remove solid fecal debris and the clear supernatantwas collected. 3 volumes of 100% ethanol with 0.1 ml of 20% sodiumacetate was added to the supernatant containing the glycoproteinfraction. Precipitation proceeded for 3 hours at room temperature. Theprecipitate was collected by centrifugation at 1040×g for 10 minutes.Traces of ethanol from the precipitate were removed by aeration. Theprecipitate was dissolved in 1 ml of Phosphate Buffered Saline (PBS).The preparation was further centrifuged at 1500×g for 15 minutes and theclear supernatant was collected.

Protein Quantitation

[0054] Protein quantitation of the samples was done by PierceBicinchoninic reagent. The assay contained (a) 100 μl each of doublingdilutions of bovine serum albumin (BioRad) ranging from 745 μg/ml, 372μg/ml, 186 μg/ml, 93 μg/ml and 46.5 μg/ml for obtaining a standardcurve; (b) 100 μl of a sample of purified COTA; and (c) 100 μl of PBS asnegative control. 2 ml of complete BCA reagent was added and tubes werecovered and incubated at RT for 2 hours. O.D. of the sample was taken at562 nm in a Shimadzu UV-160 spectrophotometer. All patient samples werediluted and normalized to 60 μg/ml protein concentration. Alternatively,protein concentrations can be obtained by spectrophotometry by adding 1ml of sample to a quartz cuvette and measuring absorbance at 280 nm.

Standard Curve of COTA

[0055] Highly purified COTA was prepared from LS174T tumors (ATCC#CL-188) by Westphals hot phenol method for complex carbohydrates(Westphal et al. Zeitschrift Fur Naturforschung 76: 148-155, 1952).Briefly, the mucinous tumor was treated with equal volume of phenol andheated at 65 C for 30 minutes. After cooling and centrifugation, theupper water soluble fraction was separated and precipitated with 3volumes of ethanol. The precipitate was dissolved in PBS, pH 7.4,dialysed and recentrifuged. It was fractionated on a Sepharose-4B columnand the fractions containing COTA reactivity were pooled andconcentrated. The preparation contained very low protein and had highCOTA reactivity. Two-fold dilutions of COTA ranging in protein values of90 μg/ml, 45 μg/ml, 22.5 μg/ml, 11.2 μg/ml and 5.6 μg/ml were used inthe dot assay and in preparation of the standard curve.

Colonoscopy

[0056] Patients were prepped for diagnostic colonoscopy by adhering to aclear liquid diet the day prior to exam, and taking an oral bowelpreparation (Co-lyte) the night prior to exam. Patients underwentcolonoscopy, the gold standard diagnostic test for colorectal neoplasia,with Olympus or Pentax video endoscopy, by board-certifiedgastroenterologists appointed to the GI laboratories of Loma LindaUniversity Medical Center or Loma Linda Veterans Administration MedicalCenter. Diagnostic information, including the size and location of anyneoplastic lesion was recorded in the procedure note, which in-turn wasforwarded to the GI research laboratory for data acquisition. Thewritten pathology report was obtained from the pathology Department,LLUMC or LLVAMC, and information was abstracted regarding tumor stage.If the patient underwent surgical resection, the operative report andfinal pathology report were reviewed to obtain tumor stage.

Slot-Dot Assay of Samples and of Dilutions of Purified COTA

[0057] O.2 μm Protan nitrocellulose membrane (Schleicher and Schuell)was placed in a Bio-Rad slot-dot apparatus. 20 μl of each test sample,known positive and negative control samples, and doubling dilutions ofpurified COTA and PBS were applied in the slots. The samples wereallowed to bind to the membrane for 30 minutes. The membrane was thenblocked with 3% non-fat milk for 30 minutes. The membrane was washedwith PBS three times and then reacted with 1 μg/ml of monoclonal SP-21antibody for 60 minutes in a humid chamber at RT. The nitrocellulosestrip was then washed with PBS and reacted with 1:2 diluted biotinylatedgoat anti-mouse antibody (Dako LSAB2 kit) and incubated for 15 minutesat RT in a humid chamber. After washing three times with PBS, themembrane was reacted with peroxidase conjugated streptavidin (DakoLSAB2) for 10 minutes at RT in a humid chamber. The membrane was washedthree times with PBS and then reacted with a solution of 5 mg/10 ml of3,3′diaminobenzidine tetrahydrochloride made in Tris buffer, pH 7.2 with0.01% H2O2 for 90 seconds. The membrane was washed with running tapwater and allowed to dry. The intensity of the slot-dot color wasquantitated by reflectance reading in a Bio-Rad model GS-700 imagingdensitometer and computer generated O.D. values were obtained. Astandard curve was drawn by plotting the COTA protein versus the opticaldensity values. From this curve, corresponding COTA equivalent proteinvamounts of the samples were calculated. Samples showing protein amountsgreater than 15 μg/ml for COTA were interpreted as positive.

Example 2 Screening for Detection of Colon Cancer Using Slot Blot Assay

[0058] On application of 20 μl of doubling dilutions of 90 μg/ml ofpurified COTA in slot-dot assay, a graduation in color intensity wasobtained (FIG. 2, lane A). Plotting the densitometer reading of eachslot-dot of purified COTA dilution against the protein amount (Table 1)resulted in a curve with 99.8% coefficient of correlation (FIG. 1).Samples with O.D. value exceeding 0.45 (15 μg/ml COTA protein) wereconsidered positive. Positive slot-dots on 3 colon cancer patients, 1ulcerative colitis and 1 polyp and negative slot-dots on 2 normal colonextracts and PBS with values lower than 15 μg/ml are seen in FIG. 2,lane B. TABLE 1 Densitometer readings of slot-dots of purified COTA COTAprotein Adjusted volume (μg/ml) O.D. (mm × mm) 90 3.09 45 1.56 22.5 0.6111.2 0.21 5.6 0.074

[0059] Sample kits were sent to 123 patients scheduled for colonoscopicexamination. 94 patients returned the sample for analysis (76.4%).Examination of 100 fecal samples for COTA levels (Table 2) consisted of94 individuals who were examined by colonoscopy and 6 healthy volunteerswho did not have colonoscopy. Of the 6 patients diagnosed with coloncancer, 5 had COTA levels higher than 15 μg/ml thus resulting in 83.3%positivity. The mean COTA value of 6 colon cancer patients was 20.8μg/ml and the mean value of 58 normal patients was 7 pg/ml. Theseresults indicate 83.3% sensitivity and 96.5% specificity of the test. Ofthe 6 patients who had prior colorectal cancers, 4 had resection of thetumors and 2 had radiation treatment of rectal cancer. Of these withpreviously treated colon cancer, one patient was positive but had 6 mmsessile polyp and had colonic obstruction at the time the fecal COTAtest was performed. Of 22 cases of polyps with polyp size ranging from 2mm to 25 mm, 6 had values over 15 μg/ml thus resulting in 27.2%positivity. Of 8 ulcerative colitis cases tested, one patient wasstrongly positive. Colonoscopy with biopsy on this patient revealed highgrade dysplasia. The group of normal patients included 6 healthy normalvolunteers. Of the remaining 52 patients who had colonoscopy done,findings include: 21 normal, 26 with diverticulosis, 4 with hemorrhoidsand one with anal fissure. A single case of hemorrhoids and anal fissuregave values in the positive range thus resulting in 3.44% positivity.These results indicate that large percentage of patients with colorectalcancer can be detected when screened for COTA in their feces.

[0060] It may be of interest to periodically follow the patients who hadearlier radiation treatment or surgery or had polyps and were stronglypositive for COTA. It is of significance that one of the ulcerativecolitis patients who had very high amounts of COTA in the feces was alsodiagnosed with high grade dysplasia (see Table 2, below). Amongindividuals who had normal colonoscopy, only 3.4% had COTA levels higherthan 15.0 μg/ml. This indicates specificity and suitability of the testfor screening colorectal neoplasms. TABLE 2 Patient samples showing COTAvalues of 15 μg/ml or higher as positives Patients No. tested No.positive No. negative % positive Colorectal CA 6 5 1 83.3 Colorectal CA6 1 5 16.6 (Resected or radiation treated) Polyps 22 6 16 27.2Ulcerative 7 0 7 12.5 colitis Ulcerative 1 1 0 100 colitis withdysplasia Normals 58 2 56 3.4

Example 3 Hybridoma Production

[0061] Monoclonal anti-COTA producing hybridomas were generated byimmunization of Balb/cj mice with highly purified COTA followed byfusion of splenocytes with SP2/0 myeloma cells. Hybridoma fluids of IgGisotype clones 13B5, 9B4, 15C11 and 15H11 and IgM isotype clones 2C3,3G4 and IBH2 have shown immunoreactivity towards COTA. The monoclonalantibodies 13B5 (IgG) and 2C3 (IgM) will be further purified by columnchromatography and protein A binding. Other monoclonal antibodies ofhybridomas (9B4 and 15C11 or 15H11) produced in our laboratory and notyet tested will be analyzed to determine if ELISA system sensitivity andspecificity can be enhanced.

Example 4 ELISA Assay for the Detection of COTA

[0062] ELISA for quantification of COTA in stool extracts will bedeveloped. The ELISA will be validated for crude stool extracts andpurified stool fractions. Twenty-four samples (in duplicate) can beprocessed in a single ELISA plate. Details of ELISA plate developmentare as follows:

[0063] 13B5 (IgG) (or alternate antibody) will be bound in the wells ofa microtiter plate as the “capture” antibody. Patient fecal samplesappropriately purified and diluted as described above will be added tothe wells. If COTA is present in the fecal sample, it is immobilized bybinding to the capture antibody. Bound COTA is then detected by theaddition of horseradish peroxidase conjugated monoclonal antibody 2C3(IgM) (or alternate antibody), and subsequent addition ofO-phenylenediamine-H202 made in citrate buffer and taking the O.D. ofthe color reaction at 492 nm. The amount of COTA in each sample isdetermined by comparison with a standard curve of COTA protein serialdilutions.

[0064] For each ELISA test plate, positive standards will include ovinesubmaxillary mucin and purified human COTA antigen in sufficientquantity as part of the ELISA kit. The standards developed will betested in a protein matrix similar to the final sample preparationprovided in the kit. Alternate ELISA configuration will be formulatedand tested. This will include binding of COTA antigen directly to theELISA 96-well plate and detecting its presence by 13B5 (IgG) and 2C3(IgM) monoclonal antibodies. To test this procedure, additional clonesIgG isotypes 9B4, 15C11 and 15H11 and IgM isotypes 3G4 and 18H2 withknown reactivity to COTA will be grown in hybridoma growth tissueculture medium and subsequently purified by liquid chromatography.

Antibody Binding to the Plate and Blocking the Reactive Sites

[0065] 100 μl of the capture antibody diluted in binding buffer to 10μg/ml is added to each well. The plate is then washed 3× with Trisbuffered saline, pH 7.6, and then blocked by adding 200 μl of Superblockto each well. The washing procedure is repeated two more times. Themicrotiter plate is used immediately in ELISA test or alternatively airdried at room temperature. The dried plates are then sealed in a ziplockplastic bag containing desiccant and stored up to 12 months at 4 C.

Antigen Bound to Plates for Testing the Presence of COTA or for TestingHybridoma Fluids

[0066] Wash plate 3 times with previously bound antigen and then blockthe remaining sites with 300 μl of blocking buffer and incubate for 30min. The plate is then washed 3 times in wash buffer. 100 μl of thefirst antibody or test antibody is diluted to proper concentration incarrier buffer and added to blanks, positive and controls (Hybridomafluids not to be diluted in carrier buffer). The plates are incubatedfor 60 min. at room temperature and then washed 3 times with washbuffer. 100 μl of second antibody diluted in carrier buffer is added toeach well and incubated for 60 min. The plate is then washed 3 timeswith wash buffer. Then 100 μl of proper substrate is added to each welland the plate is incubated for 30-60 min. at room temperature. Theabsorbance is then read using a plate reader with wavelength setaccording to type of enzyme-linked second antibody and substrate used.

Data Analysis

[0067] COTA levels expressed in micrograms per ml stool sample will bedetermined by ELISA. Levels will be correlated with the clinical gradeand stage of any neoplasm present, i.e. pre-cancerous polyp,non-invasive or invasive colon cancer, and with the disease location,i.e.: rectosigmoid, descending, transverse or ascending colon. Testsensitivity and specificity will be determined for disease stage andlocation. The positive predictive value of the COTA immunology testcompared with paired hemocult testing will be reported.

What is claimed is:
 1. A method for extracting glycoproteins from afecal sample such that immunogenicity is maintained comprising the stepsof: (a) obtaining a fecal sample from an individual; (b) shaking thefecal sample in a preservative solution; (c) separating the solutioncontaining the fecal sample to produce a fraction comprisingglycoproteins; (d) precipitating the glycoproteins from the fractioncomprising glycoproteins; and (e) dissolving the precipitatedglycoproteins in buffer.
 2. The method of claim 1 further comprising thesteps of: (f) centrifuging the solution from step (e) to produce apellet and a supernatant; and (g) collecting the supernatant containingthe extracted glycoproteins.
 3. The method of claim 1 wherein the fecalsample is collected in a clean vial containing preservative wherein thepreservative comprises ethanol and formalin at a concentration such thatbacterial growth is retarded and extraneous fecal matter is precipitatedwhile maintaining immunogenicity of glycoproteins in the fecal sample.4. The method of claim 3 wherein the preservative comprises 25-45%ethanol with 0.025%-0.35% formalin.
 5. The method of claim 4 wherein thepreservative comprises 40% ethanol with 0.25% formalin.
 6. The method ofclaim 1 wherein the solution containing the fecal sample is separated bycentrifugation.
 7. The method of claim 6 wherein the centrifugation isat 1040-1500×g for 10-15 minutes at room temperature.
 8. The method ofclaim 1 wherein the glycoproteins are precipitated from the fractioncomprising glycoproteins with 3 volumes of 100% ethanol with 0.1 ml of20% sodium acetate.
 9. The method of claim 8 wherein the precipitationproceeds for about 3 hours at room temperature.
 10. The method of claim1 wherein the precipitated glycoproteins are dissolved in phosphatebuffered saline.
 11. A method for screening for colon cancer comprising:(a) obtaining purified fecal glycoproteins, said glycoproteins beingobtained by a method comprising: (i) obtaining a fecal sample from anindividual; (ii) shaking the fecal sample in a preservative solution;(iii) separating the solution containing the fecal sample to produce afraction comprising glycoproteins; (iv) precipitating the glycoproteinsfrom the fraction comprising glycoproteins; and (v) dissolving theprecipitated glycoproteins in buffer; and (b) determining the level ofCOTA antigen in the purified fecal glycoproteins.
 12. The method ofclaim 11 wherein the fecal sample is collected in a clean vialcontaining preservative wherein the preservative comprises ethanol andformalin at a concentration such that bacterial growth is retarded andextraneous fecal matter is precipitated while maintaining immunogenicityof glycoproteins in the fecal sample.
 13. The method of claim 12 whereinthe preservative comprises 25-45% ethanol with 0.025%-0.35% formalin.14. The method of claim 13 wherein the preservative comprises 40%ethanol with 0.25% formalin.
 15. The method of claim 11 wherein thesolution containing the fecal sample is separated by centrifugation. 16.The method of claim 15 wherein centrifugation is at 1040-1500×g for10-15 minutes at room temperature.
 17. The method of claim 11 whereinthe glycoproteins are precipitated from the fraction comprisingglycoproteins with 3 volumes of 100% ethanol with 0.1 ml of 20% sodiumacetate.
 18. The method of claim 17 wherein the precipitation proceedsfor about 3 hours at room temperature.
 19. The method of claim 11wherein the precipitated glycoproteins are dissolved in phosphatebuffered saline.
 20. A method according to claim 11 whereindetermination of the level of COTA antigen in the purified glycoproteinscomprises the steps of: (a) reacting an antibody for COTA antigen withthe extracted glycoproteins to form a complex of the antibody and theCOTA antigen; (b) exposing the complex to a second antibody, whereinsaid second antibody is a detection agent; and (c) determining the levelof the detection agent and in turn determining the presence of COTAantigen in the fecal sample.
 21. The method of claim 20 wherein theantibody for COTA antigen is bound to a solid surface.
 22. The method ofclaim 20 wherein the extracted glycoproteins are bound to a solidsurface.
 23. The method of claim 20 wherein the antibody for COTAantigen is monoclonal antibody SP-21.
 24. A kit for for screening forcolon cancer comprising: an anti-COTA capture antibody bound to a solidsupport; purified human COTA antigen; and a vial containing apreservative solution.
 25. The kit of claim 24 wherein the solid supportis an ELISA plate.
 26. The kit of claim 24 wherein the solid support isa membrane filter.
 27. The kit of claim 24 wherein the antibody for COTAantigen is monoclonal antibody SP-21.
 28. The kit of claim 24 whereinthe preservative comprises 25-45% ethanol with 0.025%-0.35% formalin.29. The kit of claim 28 wherein the preservative comprises 40% ethanolwith 0.25% formalin.